Blocking Huntingtin Toxicity by Intrabody

通过体内阻断亨廷顿蛋白毒性

• 批准号: 6957329
• 负责人: XIAO-JIANG LI
• 金额: $ 19.13万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-03 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6957329
• 关键词: Huntington' s disease; antigen antibody reaction; blocking antibody; cell line; gene expression; genetically modified animals; green fluorescent proteins; homopeptide; immunocytochemistry; immunoprecipitation; laboratory mouse; monoclonal antibody; nerve /myelin protein; neuropathology; neuroprotectants; protein binding; protein folding; protein protein interaction; terminal nick end labeling; transfection /expression vector

DESCRIPTION (provided by applicant): Huntington disease (HD) belongs to a neurodegeneration disease family in which selective neuronal loss is caused by misfolded proteins. In HD, a polyglutamine (polyQ) expansion (>37 glutamines) in the N-terminal region of Huntington (htt) causes N-terminal htt fragments to misfold or aggregate, conferring neuropathology. Consistently, N-terminal htt fragments containing an expanded polyQ are able to interact with a number of proteins to mediate multiple pathological pathways. Thus, an important therapy approach is to inhibit the expression of mutant Huntington (htt) or its activity by blocking its abnormal interactions with other important proteins. Intracellular antibody (intrabody) provides a promising approach to achieve this goal, as intrabody is able to interact with an antigen intracellular to block its activity ortoxicity. However, normal htt is required for cell survival, and deletion of htt causes cell degeneration. Thus, a challenge for using the intrabody therapy for HD is to identify an intrabody that can selectively inhibit the toxicity of mutant htt but not interfere with the pivotal function of normal htt. Using the same antigen for an antibody (EM48) that reacts preferentially with misfolded and aggregated htt, we have established a hybridoma cell line that generates a monoclonal antibody (mEM48), which also preferentially reacts with mutant htt. By isolating the gene encoding mEM48 from this cell line, we have generated an intrabody that selectively binds mutant htt. We propose to characterize the protective effect of this intrabody on htt's toxicity in HD cellular models. We will also modify this intrabody to increase its expression level and stability. Finally, we will deliver this intrabody to HD mouse brain via viral vectors and to examine its protection against the neuropathology in HD brain. This proof of principle study will have a broad implication for the treatment of other neurodegeneration diseases that are also caused by misfolded proteins.

描述（由申请人提供）： 亨廷顿病 (HD) 属于神经退行性疾病家族，其中选择性神经元损失是由错误折叠的蛋白质引起的。在 HD 中，亨廷顿 (htt) N 末端区域的聚谷氨酰胺 (polyQ) 扩增（> 37 个谷氨酰胺）导致 N 末端 htt 片段错误折叠或聚集，从而产生神经病理学。一致地，含有扩展的polyQ的N端htt片段能够与许多蛋白质相互作用以介导多种病理途径。因此，一种重要的治疗方法是通过阻断突变亨廷顿蛋白（htt）与其他重要蛋白的异常相互作用来抑制其表达或其活性。胞内抗体（胞内抗体）为实现这一目标提供了一种有前途的方法，因为胞内抗体能够与胞内抗原相互作用以阻断其活性或毒性。然而，正常的htt是细胞生存所必需的，htt的缺失会导致细胞变性。因此，使用体内疗法治疗 HD 的一个挑战是鉴定一种可以选择性抑制突变 htt 毒性但不干扰正常 htt 关键功能的体内抗体。使用优先与错误折叠和聚集的 htt 反应的抗体 (EM48) 相同的抗原，我们建立了一种杂交瘤细胞系，可产生单克隆抗体 (mEM48)，该抗体也优先与突变 htt 反应。通过从该细胞系中分离编码 mEM48 的基因，我们生成了选择性结合突变体 htt 的胞内抗体。我们建议在 HD 细胞模型中表征这种体内抗体对 htt 毒性的保护作用。我们还将修改该胞内抗体以提高其表达水平和稳定性。最后，我们将通过病毒载体将这种体内递送至 HD 小鼠大脑，并检查其对 HD 大脑神经病理学的保护作用。这项原理证明研究将对治疗同样由错误折叠蛋白质引起的其他神经退行性疾病产生广泛的影响。